Compounds which inhibit rna polymerase, compositions including such compounds, and their use

ABSTRACT

RNA polymerase I (Pol I) is a dedicated polymerase for the transcription of the 47S ribosomal RNA precursor subsequently processed into the mature 5.8S, 18S and 28S ribosomal RNAs and assembled into ribosomes in the nucleolus. Pol I activity is commonly deregulated in human cancers. Based on the discovery of lead molecule BMH-21, a series of pyridoquinazolinecarboxamides were synthesized as inhibitors of Pol I and activators of the destruction of RPA194, the Pol I large catalytic subunit protein. The present invention identifies a set of bioactive compounds, including purified stereoisomers, that potently cause RPA194 degradation that function in a tightly constrained chemical space. Pharmaceutical compositions comprising these compounds and their uses in cancer and other Pol I related diseases is also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/126,588, filed on Sep. 16, 2016, which is a 35 U.S.C. § 371 U.S.national entry of International Application No. PCT/US2015/021699,having an international filing date of Mar. 20, 2015, which claims thebenefit of U.S. Provisional Application No. 61/968,079, filed Mar. 20,2014, and U.S. Provisional Application No. 62/062,197, filed Oct. 10,2014, the content of each of the aforementioned applications is hereinincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Ribosomal (r) DNA is the most highly transcribed genomic region of thehuman genome and occurs in a dedicated subcellular compartment, thenucleolus. Transcription of rRNA is mediated by RNA polymerase I (Pol I)that transcribes the multicopy rDNA gene to a long 47S rRNA precursor.The 47S rRNA precursor is processed through multiple steps to the 18S,5.8S and 28S mature rRNAs requisite for the assembly of the ribosomes.Pol I transcription is initiated by binding of a multisubunitpreinitiation complex to rDNA promoter, which stochastically recruitsthe Pol I holocomplex. The Pol I holocomplex is composed of 14 subunitsin eukaryotes, of which the subunits RPA194, RPA135 and RPA12 form thecatalytically active site. Destabilization of the rDNA helix, or loss ofthe protein framework, will effectively stall transcription. The rate ofrRNA transcription is tightly controlled by external signaling pathwaysthat cause the assembly and binding of the preinitiation complex.Deregulation of rRNA synthesis is highly frequent in human cancers. Thisis due to activation of extracellular and intracellular signalingpathways and oncogenes such as Myc. Conversely, loss-of-function oftumor suppressors p53, pRB, ARF and PTEN lead to activation of Pol Itranscription. Therefore, inhibitors of Pol I transcription may providenovel approaches toward cancer therapies.

Despite the key impact of Pol I contributing to cancer cellcharacteristics, its therapeutic exploitation has been minimal. Thepresent inventors have recently presented the discovery of an anticancersmall molecule, 12H-Benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide,N-[2(dimethylamino)ethyl]-12-oxo (BMH-21) with a distinct mode ofinhibition of Pol I compared to CX-5461 U.S. patent application Ser. No.12/665,473, filed Mar. 1, 2010 (FIG. 1). These studies demonstrated thatBMH-21 intercalates with GC-rich rDNA, inhibits Pol I and causesproteasome-mediated degradation of RPA194. BMH-21 also showed broad andpotent anticancer activity in NCI60 cancer cell lines and reduced tumorburden in mouse xenograft assays. These studies have providedproof-of-principle confirmation that Pol I targeting is a feasibleapproach for cancer control.

SUMMARY OF THE INVENTION

In accordance with an embodiment, the present invention provides acompound of formula I:

wherein X is NR₂;

wherein L is R₃ or an optionally substituted cycloamine

wherein R₁ is a straight-chained or branched C₁-C₆ hydrocarbon group(e.g., an alkyl group, an alkenyl group, an alkynyl group, alkylolgroup, hydroxyalkyl group, alkoxy group, alkoxyalkyl group, cyclicgroups, whether substituted or unsubstituted, such as cyclopentyl,cyclohexyl, pyramido, phenyl, or benzyl, cycloalkyl, heterocyclyl,indole, wherein each of alkyl, aryl, or heterocyclyl moiety may beunsubstituted or substituted with one or more substituents selected fromthe group consisting of halo, hydroxy, carboxy, phosphoryl, phosphonyl,phosphono C₁-C₆ alkyl, carboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkyl,dicarboxy halo C₁-C₆ alkyl, sulfonyl, cyano, nitro, alkoxy, alkylthio,acyl, acyloxy, thioacyl, acylthio, aryloxy, amino, alkylamino, dialkylamino, trialkylamino, arylalkylamino, guanidino, aldehydo, ureido,and aminocarbonyl, a branched or straight-chain alkylamino,dialkylamino, or alkyl or dialkylaminoalkyl, or thioalkyl, thioalkenyl,thioalkynyl, aryloxy, acyloxy, thioacyl, amido, sulphonamido, etc.), orthe like;

when X is NR₂, R₂ is H or a straight-chained C₁-C₆ alkyl group;

when L is R₃, R₃ is a straight-chained or branched C₂-C₆ alkyl group;

when L is

m=1-8 and each Y is independently selected from (CH₂)_(n)Y¹ _(p) whereinn=1-8, p=0-4 and the sum of n and p is at least 2, and each Y¹ isindependently selected from NR₄, O, S, or P, wherein R₄ is ashereinbefore defined for R₃, and X≠O; or a pharmaceutically acceptablesalt, solvate, stereoisomer, or a prodrug thereof.

In accordance with an embodiment, the present invention provides achirally pure stereoisomer of compound of formula I, wherein L is R₃ andR₃ is a straight-chained or branched C₂-C₆ alkyl group having at leastone chiral carbon.

In accordance with another embodiment, the present invention providescompounds of formula II,

wherein R₁=H and R₂=C₁-C₆ alkyl, substituted with one or more C₁-C₄alkyl, OH, NH₂, NR₃R₄, cyano, SO₂R₃, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl (including but not limited toimidazolyl, imidazolidinonyl, pyridyl, indolyl, oxazolyl, thiazolyl,oxadiazolyl), substituted or unsubstituted cycloalkyl or substituted orunsubstituted nitrogen-containing heterocycles including but not limitedto azetidine, pyrrolidine, piperidine, piperazine, azapine, morpholino;wherein R₃ and R₄, are independently selected from the group includingH, C₁-C₆ alkyl, and C₁-C₄ alkoxyl alkyl, having at least one chiralcarbon, when R₂ is substituted with at least one NR₃R₄ group.

In accordance with another embodiment, the present invention provides apharmaceutical composition comprising a compound of formula I and/orformula II, and a pharmaceutically acceptable carrier.

In accordance with a further embodiment, the present invention providesa method for activating upstream p53 pathways in a mammalian cellcomprising contacting a cell or population of cells with a compound offormula I and/or formula II.

In accordance with still another embodiment, the present inventionprovides a method for modulating RNA Pol I activity in a mammalian cellcomprising contacting a cell or population of cells with a compound offormula I and/or formula II.

In accordance with yet a further embodiment, the present inventionprovides a method for treating cancer in a subject comprisingadministering to the subject a pharmaceutical composition comprising acompound of formula I and/or formula II.

In accordance with an embodiment, the present invention provides amethod for treating cancer in a subject comprising administering to thesubject a pharmaceutical composition comprising a compound of formula Iand/or formula II, and at least one other biologically active agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the RNA Pol I inhibitor CX-5461, the BMH-21 parentmolecule, and its inactive analogue BMH-21a.

FIG. 2 shows the effect of compounds on expression and localization ofRPA194 and NCL. Immunofluorescence staining of U2OS cells treated withthe indicated compounds (0.5 μM) for 3 hours. Cells were stained for (A)RPA194 (red) and (B) NCL (green) and counterstained for DNA (blue).Scale bars, 10 μm.

FIG. 3A and FIG. 3B depict quantitative image analysis of expression andlocalization of RPA194 and NCL, respectively, by derivatives. U2OS cellswere treated with the compounds at 0, 0.1, 0.5, 1 and 5 μM and incubatedfor 3 hours. Cells were fixed and stained for (A) RPA194 and (B) NCL andcounterstained for DNA and imaged using epifluorescence. Quantitativeimage analysis for RPA194 degradation (FIG. 3A) and loss of NCLnucleolar intensity (FIG. 3B) was conducted based on two biologicalreplicates and the fold change to control is shown. Error bars, s.e.m.

FIG. 4 shows protein expression analyses for RPA194 and NCL. U2OS cellswere treated with the compounds at 0, 0.1, 1 and 10 μM and incubated for3 h. Protein was extracted using RIPA lysis buffer and Western blottingfor was conducted for RPA194, NCL and GAPDH as control.

FIG. 5 depicts cell viability assays. U2OS cells were treated with thecompounds at 0, 0.5, and 5 μM and incubated for 48 hours. Cell viabilitywas determined using WST-1 assay. N=2 biological repeats, error barss.e.m.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one or more embodiments of the present invention, aseries of BMH-21 variants were prepared and evaluated as potential novelanticancer agents that act via the repression of Pol I activity. Theactivity of BMH-21 is due to its ability to intercalate to GC-rich rDNAsequences, which makes it very different from other 4-ringanthracyclines, which cause DNA damage. Their intercalation modalitiesare also quite distinct from the anthracyclines intercalatingperpendicular to the DNA helix, whereas BMH-21 intercalates in anear-parallel fashion. While previous modeling has suggested somemolecular determinants for this activity, the high sensitivity to thependant BMH-21 chain as exemplified in the compounds identified herein,suggests there may be other components to the BMH-21-DNA complex thatlead to its biological activity. Notably, all near equipotentderivatives retained a predicted protonation of the terminal amine andhad a basic pKa close to that of the parent at 8.6. These findingsindicated that the overall charge of the inventive molecules wascritical as well as maintaining the length and basic charge close to theend of the carboxamide arm. Without being limited to any particulartheory or mechanism of action, these findings suggest that BMH-21intercalates with acidic DNA through electrostatic interactions. It alsoraises the possibility that derivatives with more highly chargedmoieties may change the nature of the intercalation or that those withlarger molecular sizes alter the DNA intercalation cavity. This furtherimplies that such molecules can perturb other DNA metabolic processes.

Therefore, in accordance with an embodiment, the present inventionprovides a compound of formula I:

wherein X is NR₂;

wherein L is R₃ or an optionally substituted cycloamine

wherein R₁ is a straight-chained or branched C₁-C₆ hydrocarbon group(e.g., an alkyl group, an alkenyl group, an alkynyl group, alkylolgroup, hydroxyalkyl group, alkoxy group, alkoxyalkyl group, cyclicgroups, whether substituted or unsubstituted, such as cyclopentyl,cyclohexyl, pyramido, phenyl, or benzyl, cycloalkyl, heterocyclyl,indole, wherein each of alkyl, aryl, or heterocyclyl moiety may beunsubstituted or substituted with one or more substituents selected fromthe group consisting of halo, hydroxy, carboxy, phosphoryl, phosphonyl,phosphono C₁-C₆ alkyl, carboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkyl,dicarboxy halo C₁-C₆ alkyl, sulfonyl, cyano, nitro, alkoxy, alkylthio,acyl, acyloxy, thioacyl, acylthio, aryloxy, amino, alkylamino, dialkylamino, trialkylamino, arylalkylamino, guanidino, aldehydo, ureido,and aminocarbonyl, a branched or straight-chain alkylamino,dialkylamino, or alkyl or dialkylaminoalkyl, or thioalkyl, thioalkenyl,thioalkynyl, aryloxy, acyloxy, thioacyl, amido, sulphonamido, etc.), orthe like;

when X is NR₂, R₂ is H or a straight-chained C₁-C₆ alkyl group;

when L is R₃, R₃ is a straight-chained or branched C₂-C₆ alkyl group;

when L is

m=1-8 and each Y is independently selected from (CH₂)_(n)Y¹ _(p) whereinn=1-8, p=0-4 and the sum of n and p is at least 2, and each Y¹ isindependently selected from NR₄, O, S, or P, wherein R₄ is ashereinbefore defined for R₃, and X≠O; or a pharmaceutically acceptablesalt, solvate, stereoisomer, or a prodrug thereof.

In accordance with an embodiment, the present invention provides achirally pure stereoisomer of compound of formula I, wherein L is R₃ andR₃ is a straight-chained or branched C₂-C₆ alkyl group having at leastone chiral carbon.

In accordance with another embodiment, the present invention providescompounds of formula II,

wherein R₁=H and R₂=C₁-C₆ alkyl, substituted with one or more C₁-C₄alkyl, OH, NH₂, NR₃R₄, cyano, SO₂R₃, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl (including but not limited toimidazolyl, imidazolidinonyl, pyridyl, indolyl, oxazolyl, thiazolyl,oxadiazolyl), substituted or unsubstituted cycloalkyl or substituted orunsubstituted nitrogen-containing heterocycles including but not limitedto azetidine, pyrrolidine, piperidine, piperazine, azapine, morpholino;wherein R₃ and R₄, are independently selected from the group includingH, C₁-C₆ alkyl, and C₁-C₄ alkoxyl alkyl, having at least one chiralcarbon, when R₂ is substituted with at least one NR₃R₄ group.

In accordance with a further embodiment, the present invention providesa chirally pure stereoisomer of compound of formula II when R₂ issubstituted with at least one NR₃R₄ group.

As used herein, examples of the term “alkyl” preferably include a C₁₋₆alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, hexyl, etc.) and the like.

As used herein, examples of the term “alkenyl” preferably include C₂₋₆alkenyl (e.g., vinyl, allyl, isopropenyl, 1-butenyl, 2-butenyl,3-butenyl, 2-methyl-2-propenyl, 1-methyl-2-propenyl,2-methyl-1-propenyl, etc.) and the like.

As used herein, examples of the term “alkynyl” preferably include C₂₋₆alkynyl (e.g., ethynyl, propargyl, 1-butynyl, 2-butynyl, 3-butynyl,1-hexynyl, etc.) and the like.

Examples of the term “aryl” preferably include a C₆₋₁₄ aryl (e.g., aphenyl, 1-naphthyl, a 2-naphthyl, 2-biphenylyl group, 3-biphenylyl,4-biphenylyl, 2-anthracenyl, etc.) and the like.

Examples of the term “arylalkyl” preferably include a C₆₋₁₄ arylalkyl(e.g., benzyl, phenylethyl, diphenylmethyl, 1-naphthylmethyl,2-naphthylmethyl, 2,2-diphenylethyl, 3-phenylpropyl, 4-phenylbutyl,5-phenylpentyl, etc.) and the like.

The term “hydroxyalkyl” embraces linear or branched alkyl groups havingone to about ten carbon atoms any one of which may be substituted withone or more hydroxyl groups.

The term “alkylamino” includes monoalkylamino. The term “monoalkylamino”means an amino, which is substituted with an alkyl as defined herein.Examples of monoalkylamino substituents include, but are not limited to,methylamino, ethylamino, isopropylamino, t-butylamino, and the like. Theterm “dialkylamino” means an amino, which is substituted with two alkylsas defined herein, which alkyls can be the same or different. Examplesof dialkylamino substituents include dimethylamino, diethylamino,ethylisopropylamino, diisopropylamino, dibutylamino, and the like.

The terms “alkylthio,” “alkenylthio” and “alkynylthio” group mean agroup consisting of a sulphur atom bonded to an alkyl-, alkenyl- oralkynyl-group, which is bonded via the sulphur atom to the entity towhich the group is bonded.

Included within the compounds of the present invention are thetautomeric forms of the disclosed compounds, isomeric forms includingdiastereoisomers, and the pharmaceutically-acceptable salts thereof.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisomericforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)-, and individual isomers are encompassed within the scope of thedisclosure. The compounds of the present invention do not include thosewhich are known in art to be too unstable to synthesize and/or isolate.The disclosure is meant to include compounds in racemic and opticallypure forms in particular attached to the R₃ substituent of compound ofFormula I. Optically active (R)- and (S)-, isomers may be prepared usingchiral synthons or chiral reagents as disclosed herein, or resolvedusing conventional techniques. When the compounds described hereincontain olefinic bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers.

The term “pharmaceutically acceptable salts” embraces salts commonlyused to form alkali metal salts and to form addition salts of free acidsor free bases. Examples of acids which may be employed to formpharmaceutically acceptable acid addition salts include such inorganicacids as hydrochloric acid, sulphuric acid and phosphoric acid, and suchorganic acids as maleic acid, succinic acid and citric acid. Otherpharmaceutically acceptable salts include salts with alkali metals oralkaline earth metals, such as sodium, potassium, calcium and magnesium,or with organic bases, such as dicyclohexylamine. Suitablepharmaceutically acceptable salts of the compounds of the presentinvention include, for example, acid addition salts which may, forexample, be formed by mixing a solution of the compound according to theinvention with a solution of a pharmaceutically acceptable acid, such ashydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid,maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid,citric acid, tartaric acid, carbonic acid or phosphoric acid. All ofthese salts may be prepared by conventional means by reacting, forexample, the appropriate acid or base with the corresponding compoundsof the present invention.

Salts formed from free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

For use in medicines, the salts of the compounds of the presentinvention should be pharmaceutically acceptable salts. Other salts may,however, be useful in the preparation of the compounds according to theinvention or of their pharmaceutically acceptable salts.

In addition, embodiments of the invention include hydrates of thecompounds of the present invention. The term “hydrate” includes but isnot limited to hemihydrate, monohydrate, dihydrate, trihydrate and thelike. Hydrates of the compounds of the present invention may be preparedby contacting the compounds with water under suitable conditions toproduce the hydrate of choice.

In accordance with one or more of the foregoing embodiments, the presentinvention provides a compound selected from the group consisting of:

In accordance with an embodiment, the present invention providespharmaceutical compositions comprising the compounds of formula I, ortheir salts, solvates, or stereoisomers thereof, and a pharmaceuticallyacceptable carrier.

Embodiments of the invention also include a process for preparingpharmaceutical products comprising the compounds. The term“pharmaceutical product” means a composition suitable for pharmaceuticaluse (pharmaceutical composition), as defined herein. Pharmaceuticalcompositions formulated for particular applications comprising thecompounds of the present invention are also part of this invention, andare to be considered an embodiment thereof.

As such, in accordance with an embodiment, the present inventionprovides a pharmaceutical composition comprising the compound of formulaI:

wherein X is NR₂;

wherein L is R₃ or an optionally substituted cycloamine

wherein R₁ is a straight-chained or branched C₁-C₆ hydrocarbon group(e.g., an alkyl group, an alkenyl group, an alkynyl group, alkylolgroup, hydroxyalkyl group, alkoxy group, alkoxyalkyl group, cyclicgroups, whether substituted or unsubstituted, such as cyclopentyl,cyclohexyl, pyramido, phenyl, or benzyl, cycloalkyl, heterocyclyl,indole, wherein each of alkyl, aryl, or heterocyclyl moiety may beunsubstituted or substituted with one or more substituents selected fromthe group consisting of halo, hydroxy, carboxy, phosphoryl, phosphonyl,phosphono C₁-C₆ alkyl, carboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkyl,dicarboxy halo C₁-C₆ alkyl, sulfonyl, cyano, nitro, alkoxy, alkylthio,acyl, acyloxy, thioacyl, acylthio, aryloxy, amino, alkylamino, dialkylamino, trialkylamino, arylalkylamino, guanidino, aldehydo, ureido,and aminocarbonyl, a branched or straight-chain alkylamino,dialkylamino, or alkyl or dialkylaminoalkyl, or thioalkyl, thioalkenyl,thioalkynyl, aryloxy, acyloxy, thioacyl, amido, sulphonamido, etc.), orthe like;

when X is NR₂, R₂ is H or a straight-chained C₁-C₆ alkyl group;

when L is R₃, R₃ is a straight-chained or branched C₂-C₆ alkyl group;

when L is

m=1-8 and each Y is independently selected from (CH₂)_(n)Y¹ _(p) whereinn=1-8, p=0-4 and the sum of n and p is at least 2, and each Y¹ isindependently selected from NR₄, O, S, or P, wherein R₄ is ashereinbefore defined for R₃, and X≠O; or a pharmaceutically acceptablesalt, solvate, stereoisomer, or a prodrug thereof, and apharmaceutically acceptable carrier, in an effective amount, for use asa medicament, preferably for use in inhibiting RNA Pol I in a mammaliancell or population of cells, or for use in treating cancer in a subject.

In accordance with an embodiment, the present invention provides apharmaceutical composition comprising chirally pure stereoisomer ofcompound of formula I, wherein L is R₃ and R₃ is a straight-chained orbranched C₂-C₆ alkyl group having at least one chiral carbon, and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition can further comprise at least one additionalbiologically active agent.

In accordance with another embodiment, the present invention provides apharmaceutical composition comprising compounds of formula II,

wherein R₁=H and R₂=C₁-C₆ alkyl, substituted with one or more C₁-C₄alkyl, OH, NH₂, NR₃R₄, cyano, SO₂R₃, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl (including but not limited toimidazolyl, imidazolidinonyl, pyridyl, indolyl, oxazolyl, thiazolyl,oxadiazolyl), substituted or unsubstituted cycloalkyl or substituted orunsubstituted nitrogen-containing heterocycles including but not limitedto azetidine, pyrrolidine, piperidine, piperazine, azapine, morpholino;wherein R₃ and R₄, are independently selected from the group includingH, C₁-C₆ alkyl, and C₁-C₄ alkoxyl alkyl, having at least one chiralcarbon, when R₂ is substituted with at least one NR₃R₄ group.

In accordance with a further embodiment, the present invention providesa pharmaceutical composition comprising a chirally pure stereoisomer ofcompound of formula II when R₂ is substituted with at least one NR₃R₄group and a pharmaceutically acceptable carrier. In some embodiments,the pharmaceutical composition can further comprise at least oneadditional biologically active agent.

In accordance with another embodiment, the present invention provides apharmaceutical composition comprising at least one of the compoundsselected from the group consisting of:

and a pharmaceutically acceptable carrier, in an effective amount, foruse as a medicament, preferably for use in inhibiting RNA Pol I in amammalian cell or population of cells, or for use in treating cancer ina subject.

With respect to pharmaceutical compositions described herein, thepharmaceutically acceptable carrier can be any of those conventionallyused, and is limited only by physico-chemical considerations, such assolubility and lack of reactivity with the active compound(s), and bythe route of administration. The pharmaceutically acceptable carriersdescribed herein, for example, vehicles, adjuvants, excipients, anddiluents, are well-known to those skilled in the art and are readilyavailable to the public. Examples of the pharmaceutically acceptablecarriers include soluble carriers such as known buffers which can bephysiologically acceptable (e.g., phosphate buffer) as well as solidcompositions such as solid-state carriers or latex beads. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active agent(s), and one which has little or nodetrimental side effects or toxicity under the conditions of use.

The carriers or diluents used herein may be solid carriers or diluentsfor solid formulations, liquid carriers or diluents for liquidformulations, or mixtures thereof.

Solid carriers or diluents include, but are not limited to, gums,starches (e.g., corn starch, pregelatinized starch), sugars (e.g.,lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g.,microcrystalline cellulose), acrylates (e.g., polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers may be,for example, aqueous or non-aqueous solutions, suspensions, emulsions oroils. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, and injectable organic esters such as ethyl oleate.Aqueous carriers include, for example, water, alcoholic/aqueoussolutions, cyclodextrins, emulsions or suspensions, including saline andbuffered media.

Examples of oils are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, mineral oil, olive oil,sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil,olive, petrolatum, and mineral. Suitable fatty acids for use inparenteral formulations include, for example, oleic acid, stearic acid,and isostearic acid. Ethyl oleate and isopropyl myristate are examplesof suitable fatty acid esters.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include, for example, sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's andfixed oils. Formulations suitable for parenteral administration include,for example, aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain anti-oxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives.

Intravenous vehicles include, for example, fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Examples are sterile liquids such as water andoils, with or without the addition of a surfactant and otherpharmaceutically acceptable adjuvants. In general, water, saline,aqueous dextrose and related sugar solutions, and glycols such aspropylene glycols or polyethylene glycol are preferred liquid carriers,particularly for injectable solutions.

In addition, in an embodiment, the compounds of the present inventionmay further comprise, for example, binders (e.g., acacia, cornstarch,gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g.,cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelosesodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g.,Tris-HCl, acetate, phosphate) of various pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., cremophor, glycerol,polyethylene glycerol, benzlkonium chloride, benzyl benzoate,cyclodextrins, sorbitan esters, stearic acids), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite, butylated hydroxyanisole),stabilizers (e.g., hydroxypropyl cellulose, hydroxypropylmethylcellulose), viscosity increasing agents (e.g., carbomer, colloidalsilicon dioxide, ethyl cellulose, guar gum), sweetners (e.g., aspartame,citric acid), preservatives (e.g., thimerosal, benzyl alcohol,parabens), lubricants (e.g., stearic acid, magnesium stearate,polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidalsilicon dioxide), plasticizers (e.g., diethyl phthalate, triethylcitrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodiumlauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines),coating and film forming agents (e.g., ethyl cellulose, acrylates,polymethacrylates), and/or adjuvants.

The choice of carrier will be determined, in part, by the particularcompound, as well as by the particular method used to administer thecompound. Accordingly, there are a variety of suitable formulations ofthe pharmaceutical composition of the invention. The followingformulations for parenteral, subcutaneous, intravenous, intramuscular,intraarterial, intrathecal and interperitoneal administration areexemplary, and are in no way limiting. More than one route can be usedto administer the compounds, and in certain instances, a particularroute can provide a more immediate and more effective response thananother route.

Suitable soaps for use in parenteral formulations include, for example,fatty alkali metal, ammonium, and triethanolamine salts, and suitabledetergents include, for example, (a) cationic detergents such as, forexample, dimethyl dialkyl ammonium halides, and alkyl pyridiniumhalides, (b) anionic detergents such as, for example, alkyl, aryl, andolefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, andsulfosuccinates, (c) nonionic detergents such as, for example, fattyamine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylenecopolymers, (d) amphoteric detergents such as, for example,alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammoniumsalts, and (e) mixtures thereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the compounds in solution. Preservatives andbuffers may be used. In order to minimize or eliminate irritation at thesite of injection, such compositions may contain one or more nonionicsurfactants, for example, having a hydrophile-lipophile balance (HLB) offrom about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include, for example, polyethylene glycol sorbitanfatty acid esters, such as sorbitan monooleate and the high molecularweight adducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol.

The parenteral formulations can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630(2009)).

For purposes of the invention, the amount or dose of the compounds,salts, solvates, or stereoisomers of any one the compounds of Formula I,as set forth above, administered should be sufficient to effect, e.g., atherapeutic or prophylactic response, in the subject over a reasonabletime frame. The dose will be determined by the efficacy of theparticular compound and the condition of a human, as well as the bodyweight of a human to be treated.

It is understood by those of ordinary skill, that the compounds of thepresent invention are inhibitors of RNA polymerase I through one or moremechanisms of action. Without being limited to any particular theory,the compounds of the present invention can inhibit RNA Pol I byintercalation of the nucleic acids at G-C rich regions which block thepolymerase activity.

One of ordinary skill in the art understands that p53 is a highlyresponsive molecule to cellular stress and DNA damage, and implicated indiverse diseases like cancer, ischemia, neuronal disorders, inflammationand also during physiological processes like in normal cellularmetabolism, development and aging. Thus, the compounds of the presentinvention are useful in prevention or treatment of diseases involvingthe p53 pathways.

Therefore, in accordance with an embodiment, the present inventionprovides the use of the compounds or the pharmaceutical compositionsdisclosed herein in an amount effective for activating upstream p53pathways in a mammalian cell comprising contacting a cell or populationof cells with a compound of formula I.

In accordance with an embodiment, the present invention provides the useof the compounds or the pharmaceutical compositions disclosed herein inan amount effective for modulating RNA Pol I activity in a mammaliancell comprising contacting a cell or population of cells with a compoundof formula I.

In accordance with an embodiment, the present invention provides the useof the compounds or the pharmaceutical compositions disclosed herein inan amount effective for treating cancer or a hyperproliferative diseasein a subject comprising administering to the subject a pharmaceuticalcomposition comprising a compound of formula I. In an alternativeembodiment, the use includes at least one additional biologically activeagent.

The dose of the compounds, salts, solvates, or stereoisomers of any onethe compounds of Formula I, as set forth above, of the present inventionalso will be determined by the existence, nature and extent of anyadverse side effects that might accompany the administration of aparticular compound. Typically, an attending physician will decide thedosage of the compound with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, compound to be administered, route ofadministration, and the severity of the condition being treated. By wayof example, and not intending to limit the invention, the dose of thecompound can be about 0.001 to about 1000 mg/kg body weight of thesubject being treated/day, from about 0.01 to about 100 mg/kg bodyweight/day, or from about 1 mg to about 100 mg/kg body weight/day. Insome embodiments the dosage of the compound can be in the range of about0.1 μM to about 100 μM, preferably about 1 μM to about 50 μM.

Alternatively, the compounds of the present invention can be modifiedinto a depot form, such that the manner in which the compound isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of compounds can be, for example, animplantable composition comprising the compound and a porous ornon-porous material, such as a polymer, wherein the compound isencapsulated by or diffused throughout the material and/or degradationof the non-porous material. The depot is then implanted into the desiredlocation within the body and the compounds are released from the implantat a predetermined rate.

In one embodiment, the compounds of the present invention providedherein can be controlled release compositions, i.e., compositions inwhich the one or more compounds are released over a period of time afteradministration. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Inanother embodiment the composition is an immediate release composition,i.e., a composition in which all, or substantially all of the compound,is released immediately after administration.

In yet another embodiment, the compounds of the present invention can bedelivered in a controlled release system. For example, the agent may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, or other modes of administration. In an embodiment, apump may be used. In one embodiment, polymeric materials can be used. Inyet another embodiment, a controlled release system can be placed inproximity to the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Design of ControlledRelease Drug Delivery Systems, Xiaoling Li and Bhaskara R. Jasti eds.(McGraw-Hill, 2006)).

The compounds included in the pharmaceutical compositions of the presentinvention may also include incorporation of the active ingredients intoor onto particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, hydrogels, etc., or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance.

In accordance with the present invention, the compounds of the presentinvention may be modified by, for example, the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Themodified compounds are known to exhibit substantially longer half-livesin blood following intravenous injection, than do the correspondingunmodified compounds. Such modifications may also increase thecompounds' solubility in aqueous solution, eliminate aggregation,enhance the physical and chemical stability of the compound, and greatlyreduce the immunogenicity and reactivity of the compound. As a result,the desired in vivo biological activity may be achieved by theadministration of such polymer-compound adducts less frequently, or inlower doses than with the unmodified compound.

An active agent and a biologically active agent are used interchangeablyherein to refer to a chemical or biological compound that induces adesired pharmacological and/or physiological effect, wherein the effectmay be prophylactic or therapeutic. The terms also encompasspharmaceutically acceptable, pharmacologically active derivatives ofthose active agents specifically mentioned herein, including, but notlimited to, salts, esters, amides, prodrugs, active metabolites, analogsand the like. When the terms “active agent,” “pharmacologically activeagent” and “drug” are used, then, it is to be understood that theinvention includes the active agent per se as well as pharmaceuticallyacceptable, pharmacologically active salts, esters, amides, prodrugs,metabolites, analogs etc. The active agent can be a biological entity,such as a virus or cell, whether naturally occurring or manipulated,such as transformed.

Further examples of biologically active agents include, withoutlimitation, enzymes, receptor antagonists or agonists, hormones, growthfactors, autogenous bone marrow, antibiotics, antimicrobial agents, RNAand DNA molecules and nucleic acids, and antibodies. Specific examplesof useful biologically active agents the above categories include:anti-neoplastics such as androgen inhibitors, antimetabolites, cytotoxicagents, and immunomodulators.

Biologically active agents also include anti-cancer agents such asalkylating agents, nitrogen mustard alkylating agents, nitrosoureaalkylating agents, antimetabolites, purine analog antimetabolites,pyrimidine analog antimetabolites, hormonal antineoplastics, naturalantineoplastics, antibiotic natural antineoplastics, and vinca alkaloidnatural antineoplastics.

Further examples of alkylating antineoplastic agents include carboplatinand cisplatin; nitrosourea alkylating antineoplastic agents, such ascarmustine (BCNU); antimetabolite antineoplastic agents, such asmethotrexate; pyrimidine analog antineoplastic agents, such asfluorouracil (5-FU) and gemcitabine; hormonal antineoplastics, such asgoserelin, leuprolide, and tamoxifen; natural antineoplastics, such asaldesleukin, interleukin-2, docetaxel, etoposide, interferon;paclitaxel, other taxane derivatives, and tretinoin (ATRA); antibioticnatural antineoplastics, such as bleomycin, dactinomycin, daunorubicin,doxorubicin, and mitomycin; vinca alkaloid natural antineoplastics, suchas vinblastine and vincristine, and PD1 inhibitors such aslambrolizumab.

As used herein, the term “subject” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

As used herein, the term “modulate” means that the compounds of formulaI, described herein either increase or decrease the activity of RNA PolI.

As used herein, the term “hyperproliferative disease” includes cancerand other diseases such as neoplasias and hyperplasias. Cellularproliferative diseases include, for example, rheumatoid arthritis,inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas,lipomas, hemangiomas, fibromas, vascular occlusion, restenosis,artherosclerosis, a pre-neoplastic lesion, carcinoma in situ, oral hairyleukoplakia, or psoriasis. In accordance with one or more embodiments,the term cancer can include any of acute lymphocytic cancer, acutemyeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer,central nervous system cancer, peripheral nerve sheet tumors, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vulva, chroniclymphocytic leukemia, chronic myeloid cancer, colon cancer, esophagealcancer, cervical cancer, gastrointestinal carcinoid tumor. Hodgkinlymphoma, hypopharynx cancer, kidney cancer, larynx cancer, livercancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma,nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreaticcancer, peritoneum, omentum, and mesentery cancer, pharynx cancer,prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma(RCC)), small intestine cancer, soft tissue cancer, stomach cancer,testicular cancer, thyroid cancer, ureter cancer, and urinary bladdercancer.

EXAMPLES

Cells and Viability Assay. The cells were maintained at 37° C. in ahumidified atmosphere containing 5% CO2. U2OS ostesarcoma cells werecultured in DMEM supplemented with 15% fetal bovine serum. Cells wereplated in 96-well plates at a density of 10,000 cells/well in triplicateand incubated for 48 hours with the compounds. Viability was determinedusing WST-1 cell proliferation reagent (Roche Diagnostics).

Immunofluorescence, Epifluorescence Microscopy and Image Analysis. U2OScells grown on coverslips were fixed in 3.5% paraformaldehyde,permeabilized with 0.5% NP-40 and blocked with 3% BSA as described inPeltonen et al (Mol. Cancer Ther. 2014). Cells were stained for RPA194(C-1, Santa Cruz Biotechnology) and NCL (4E2, Abcam). Alexa 488 andAlexa 594-conjugated anti-mouse or anti-rabbit antibodies were fromInvitrogen. DNA was counterstained with DAPI (Invitrogen). Images werecaptured using Axioplan2 fluorescence microscope (Zeiss) equipped withAxioCam HRc CCD-camera and AxioVision 4.5 software using ECPlan-Neofluar 20×/0.75 objective (Zeiss). Images were quantified usingFrIDA image analysis software as described in Peltonen et al. (2014).Hue saturation and brightness range were defined individually for RPA194and NCL. All values were normalized to the DNA content. Two-four fieldsof each treatment were recorded and quantification was based on anaverage of 200 cells.

Immunoblotting. Lysis of cells was conducted in 0.5% NP-40 buffer (25 mMTris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40, 4 mM NaF, 100 μM Na₃VO₄, 100KIU/mL aprotinin, 10 μg/mL leupeptin). Proteins were separated onSDS-PAGE gel and blotted as in (Peltonen et al. 2010). The followingantibodies were used: NCL (4E2, Abeam), RPA194 (C-1, Santa CruzBiotechnology), GAPDH (Europa Bioproducts). HRP-conjugated secondaryantibodies were from DAKO.

Determination of RPA194 and NCL IC₅₀. U2OS cells grown on coverslipswere treated the compounds at 0.1, 0.5, 1, 5 and 10 or vehicle(DMSO)-treated for 3 h and fixed and stained as above. All compounds,except when indicated, were tested in duplicate independent biologicalexperiments. Immunostaining for NCL and RPA194 was conducted separately,and cells were counterstained for DNA. Two-four fields of each treatmentwere captured using epifluorescence microscopy as above, and containedon average of 200 cells/analysis. The images were quantified using FrIDAimage analysis software as described in ref. 14. Hue saturation andbrightness range were defined individually for RPA194 and NCL, and allvalues were normalized to the DNA content. The fold change to controlwas determined. IC₅₀ was determined by GraphPad Prism for Windows(version 6.01) using a three-parameter fit.

Synthesis. General Methods. All commercially available reagents andsolvents were used without further purification unless otherwise stated.Automated flash chromatography was performed on an ISCO CombiFlash Rf orBiotage Isolera using Biotage Flash cartridges with peak detection at254 nm. Reverse phase purification was accomplished using a Gilson 215liquid handler equipped with a Phenomenex C18 column (150×20 mm I.D.,S-5 μm). Peak collection was triggered by UV detection at 214 or 254 nm.¹H NMR spectra were recorded on a Bruker 400 instrument operating at 400MHz with tetramethylsilane or residual protonated solvent used as areference. Analytical LC/MS was performed using Agilent 1260 equippedwith autosampler (Agilent Poroshell 120 C18 column (50×4.6 mm I.D., 3.5μm); 0.1% TFA in water/acetonitrile gradient; UV detection at 215 and254 nm) and electrospray ionization.

The general synthetic scheme:

12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxylic acid (3a). Amixture of 3-amino-2-naphthoic acid (2) (2.00 g, 10.68 mmol),2-chloronicotinic acid (1) (1.68 g, 10.68 mmol) and hydrochloric acid(0.9 mL, 29.13 mmol) in ethanol (70 mL) were stirred at 80° C. for 66 h(for convenience). After cooling, the reddish-orange suspension wasfiltered, washed with ethanol and air-dried to give12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxylic acid (1.56 g,5.37 mmol, 50.3% yield) as a yellow-orange solid. ¹H NMR (400 MHz,DMSO-d6) δ ppm 9.16 (s, 1H) 9.03 (dd, J=7.20, 1.64 Hz, 1H) 8.63 (dd,J=6.95, 1.64 Hz, 1H) 8.49 (s, 1H) 8.34 (d, J=8.34 Hz, 1H) 8.19 (d,J=8.08 Hz, 1H) 7.76 (t, J=7.07 Hz, 1H) 7.64 (t, J=6.95 Hz, 1H) 7.18 (t,J=7.07 Hz, 1H). MS [M+1]=291.

11-oxopyrido[2,1-b]quinazoline-6-carboxylic acid (3b). A mixture of2-aminobenzoic acid (250 mg, 1.82 mmol), 2-chloronicotinic acid (287.2mg, 1.82 mmol) and hydrochloric acid (0.3 mL, 9.85 mmol) in ethanol (20mL) were stirred at 80° C. for 48 h (for convenience). After cooling,the reddish-orange suspension was filtered, washed with ethanol andair-dried to give 11-oxopyrido[2,1-b]quinazoline-6-carboxylic acid (107mg, 0.45 mmol, 24.5% yield) as a pale yellow solid. ¹H NMR (400 MHz,CDCl₃) δ ppm 9.50 (dd, J=7.07, 1.52 Hz, 3H) 9.18 (dd, J=7.45, 1.64 Hz,3H) 8.56 (dd, J=8.21, 1.39 Hz, 3H) 8.17 (ddd, J=8.46, 7.20, 1.52 Hz, 3H)8.03 (s, 2H) 8.01 (s, 1H) 7.80 (ddd, J=8.15, 7.26, 1.01 Hz, 4H) 7.72 (t,J=7.20 Hz, 3H). MS [M+1]=241.

Method A: Synthesis of amide analogues (4).N-[2-(dimethylamino)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide.To a solution of12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxylic acid (50. mg,0.17 mmol) and TBTU (82.9 mg, 0.26 mmol) in DMF (1 mL) was added DIPEA(90 μL, 0.52 mmol). After the contents were stirred at room temperaturefor 15 minutes, N,N-dimethylethylenediamine (28.4 μL, 0.26 mmol) wasadded, and stirring continued for 16 hours (for convenience). Addedreaction mixture to 100 mL cold water with stirring. Collected solid byfiltration and dried under vacuum to giveN-[2-(dimethylamino)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(36 mg, 0.10 mmol, 58.0% yield) as a yellow solid. 1H NMR (400 MHz,DMSO-d6) δ ppm 11.50 (br. s., 1H) 9.10 (s, 1H) 8.91 (d, J=5.81 Hz, 1H)8.55 (d, J=5.56 Hz, 1H) 8.28-8.34 (m, 2H) 8.12 (d, J=8.34 Hz, 1H) 7.73(t, J=7.45 Hz, 1H) 7.61 (t, J=7.33 Hz, 1H) 7.05 (t, J=7.07 Hz, 1H) 3.56(d, J=5.05 Hz, 2H) 2.59 (t, J=5.94 Hz, 2H) 2.40 (s, 6H). 1H NMR (400MHz, CDCl3) δ ppm 11.70 (br. s., 1H) 9.10 (s, 1H) 8.94 (dd, J=7.33, 1.77Hz, 1H) 8.73 (dd, J=6.82, 1.77 Hz, 1H) 8.29 (s, 1H) 8.12 (d, J=8.59 Hz,1H) 8.00 (d, J=8.34 Hz, 1H) 7.66 (t, J=7.58 Hz, 1H) 7.52-7.60 (m, 1H)6.89 (t, J=7.07 Hz, 1H) 3.66-3.77 (m, 2H) 2.71 (t, J=6.06 Hz, 2H) 2.49(s, 6H). MS [M+1]=361.

Example 1

N-[2-(dimethylamino)ethyl]-N-methyl-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 1). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N,N,N′-trimethylethylenediamine (21.3 μL, 0.17 mmol) according toMethod A to giveN-[2-(dimethylamino)ethyl]-N-methyl-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamidetrifluoroacetate (18.8 mg, 0.050 mmol, 29.1% yield) as an orange solid.¹H NMR (400 MHz, CDCl₃) δ ppm 9.14 (s, 1H) 8.93 (d, J=7.33 Hz, 1H) 8.28(s, 1H) 8.14 (d, J=8.34 Hz, 1H) 8.04 (d, J=8.84 Hz, 1H) 7.93 (d, J=6.82Hz, 1H) 7.67-7.74 (m, 1H) 7.58-7.64 (m, 1H) 6.97 (t, J=7.07 Hz, 1H) 4.12(t, J=5.94 Hz, 2H) 3.57 (t, J=6.19 Hz, 2H) 3.12 (s, 6H) 3.10 (s, 3H). MS[M+1]=375.

Example 2

N-[2-(dimethylamino)ethyl]-N-ethyl-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 2). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N,N-dimethyl-N′-ethylethylenediamine (27.1 μL, 0.17 mmol) accordingto Method A to giveN-[2-(dimethylamino)ethyl]-N-ethyl-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamidetrifluoroacetate (36.1 mg, 0.093 mmol, 54% yield) as a yellow-orangesolid. ¹H NMR (400 MHz, CDCl₃) δ ppm 9.13 (s, 1H) 8.87 (d, J=7.33 Hz,1H) 8.21 (s, 1H) 8.13 (d, J=8.34 Hz, 1H) 8.03 (d, J=8.59 Hz, 1H)7.65-7.71 (m, 1H) 7.56-7.62 (m, 2H) 6.84 (t, J=7.07 Hz, 1H) 4.06 (m, 2H)3.62 (t, J=6.69 Hz, 2H) 3.39 (q, J=7.24 Hz, 2H) 3.12 (s, 6H) 1.20 (t,J=7.07 Hz, 3H). MS [M+1]=389.

Example 3

N-(2-imidazol-1-ylethyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 3). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 2-imidazol-1-ylethanamine (16.6 μL, 0.17 mmol) according to Method Ato giveN-(2-imidazol-1-ylethyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(28.5 mg, 0.074 mmol, 43% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 11.57 (br. s., 1H) 9.08 (s, 1H) 8.98 (dd, J=7.07, 1.77Hz, 1H) 8.73 (dd, J=7.07, 1.77 Hz, 1H) 8.10 (t, J=8.34 Hz, 3H) 7.94 (s,1H) 7.66-7.72 (m, 3H) 7.55-7.60 (m, 2H) 7.22 (s, 1H) 7.16 (s, 1H) 6.91(t, J=7.07 Hz, 2H) 4.32-4.37 (m, 2H) 4.01-4.07 (m, 2H). MS [M+1]=384.

Example 4

12-oxo-N-[2-(2-pyridyl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 4). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 2-(2-aminoethyl)pyridine (20.6 μL, 0.17 mmol) according to Method Ato give12-oxo-N-[2-(2-pyridyl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(40.7 mg, 0.103 mmol, 60% yield) as an orange solid. ¹H NMR (400 MHz,DMSO-d6) δ ppm 11.39 (br. s., 1H) 9.12 (s, 1H) 8.96 (dd, J=7.33, 1.77Hz, 1H) 8.75 (dd, J=7.07, 1.77 Hz, 1H) 8.26 (s, 1H) 8.13 (d, J=8.59 Hz,1H) 8.03 (d, J=8.59 Hz, 1H) 7.64-7.72 (m, 1H) 7.53-7.61 (m, 1H) 6.91 (t,J=7.07 Hz, 1H) 3.64-3.74 (m, 2H) 2.50-2.58 (m, 2H). MS [M+1]=395.

Example 5

12-oxo-N-[2-(3-pyridyl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 5). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 3-(2-aminoethyl)pyridine (20.5 μL, 0.17 mmol) according to Method Ato give12-oxo-N-[2-(3-pyridyl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(55 mg, 0.14 mmol, 81% yield) as an orange solid. ¹H NMR (400 MHz,DMSO-d6) δ ppm 11.39 (br. s., 1H) 9.12 (s, 1H) 8.96 (dd, J=7.33, 1.77Hz, 1H) 8.75 (dd, J=7.07, 1.77 Hz, 1H) 8.26 (s, 1H) 8.13 (d, J=8.59 Hz,1H) 8.03 (d, J=8.59 Hz, 1H) 7.64-7.72 (m, 1H) 7.53-7.61 (m, 1H) 6.91 (t,J=7.07 Hz, 1H) 3.64-3.74 (m, 2H) 2.50-2.58 (m, 2H). MS [M+1]=395.

Example 6

12-oxo-N-[2-(2-oxoimidazolidin-1-yl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 6). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 1-(2-aminoethyl)imidazolidin-2-one (19.6 μL, 0.17 mmol) according toMethod A to give12-oxo-N-[2-(2-oxoimidazolidin-1-yl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(6.5 mg, 0.016 mmol, 9.4% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 11.54 (br. s., 1H) 9.12 (s, 1H) 8.98 (dd, J=7.20, 1.64Hz, 1H) 8.74 (d, J=7.07 Hz, 1H) 8.43 (s, 1H) 8.12 (dd, J=12.63, 8.08 Hz,2H) 7.66-7.71 (m, 1H) 7.57 (dd, J=15.79, 8.72 Hz, 2H) 7.02 (s, 1H) 6.91(t, J=7.33 Hz, 1H) 3.80-3.88 (m, 2H) 3.61-3.69 (m, 4H) 3.47 (m, 2H). MS[M+1]=402.

Example 7

N-[2-(1H-indol-3-yl)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 7). This compound was synthesized from 3a (50 mg, 0.17 mmol)and tryptamine (27.6 mg, 0.17 mmol) according to Method A to giveN-[2-(1H-indol-3-yl)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(22.9 mg, 0.053 mmol, 30.7% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 11.43-11.50 (m, 1H) 9.04 (s, 1H) 8.91-8.95 (m, 1H)8.77 (dd, J=6.82, 1.77 Hz, 1H) 8.06 (s, 2H) 7.83 (d, J=7.33 Hz, 1H) 7.74(d, J=8.08 Hz, 1H) 7.60-7.67 (m, 1H) 7.54 (s, 1H) 7.47 (s, 1H) 7.34 (s,2H) 7.19-7.26 (m, 3H) 7.16 (d, J=2.02 Hz, 2H) 6.90 (t, J=7.20 Hz, 1H)4.08 (d, J=6.06 Hz, 2H) 3.27 (m, 2H). MS [M+1]=433.

Example 8

N-(2-methylsulfonylethyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 8). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 2-methylsulfonylethanamine (21.2 mg, 0.17 mmol) according to MethodA to giveN-(2-methylsulfonylethyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(2.1 mg, 0.0053 mmol, 3.1% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl3) δ ppm 11.93 (br. s., 1H) 9.12 (s, 1H) 9.00 (dd, J=7.45, 1.64Hz, 1H) 8.73 (dd, J=6.95, 1.64 Hz, 1H) 8.49 (s, 1H) 8.12 (t, J=8.84 Hz,2H) 7.67-7.72 (m, 1H) 7.56-7.62 (m, 1H) 6.92 (t, J=7.07 Hz, 1H)4.16-4.22 (m, 2H) 3.49-3.54 (m, 2H) 3.07 (s, 3H). MS [M+1]=396.

Example 9

N-[3-(dimethylamino)propyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 9). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N,N-dimethyl-1,3-propanediamine (21.7 μL, 0.17 mmol) according toMethod A to giveN-[3-(dimethylamino)propyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(18.1 mg, 0.048 mmol, 28.1% yield) as an orange solid. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 11.39 (br. s., 1H) 9.12 (s, 1H) 8.96 (dd, J=7.33, 1.77Hz, 1H) 8.75 (dd, J=7.07, 1.77 Hz, 1H) 8.26 (s, 1H) 8.13 (d, J=8.59 Hz,1H) 8.03 (d, J=8.59 Hz, 1H) 7.64-7.72 (m, 1H) 7.53-7.61 (m, 1H) 6.91 (t,J=7.07 Hz, 1H) 3.64-3.74 (m, 2H) 2.50-2.58 (m, 2H) 2.33 (s, 6H) 1.98(quin, J=7.14 Hz, 2H). MS [M+1]=375.

Example 10

N-[4-(dimethylamino)butyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 10). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N′,N′-dimethylbutane-1,4-diamine (24.5 μL, 0.17 mmol) according toMethod A to giveN-[4-(dimethylamino)butyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(22.1 mg, 0.057 mmol, 33% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 11.35-11.41 (m, 1H) 9.13 (s, 1H) 8.98 (dd, J=7.20,1.64 Hz, 1H) 8.76 (dd, J=6.95, 1.64 Hz, 1H) 8.26 (s, 1H) 8.14 (d, J=8.59Hz, 1H) 8.06 (d, J=8.59 Hz, 1H) 7.66-7.72 (m, 1H) 7.54-7.63 (m, 1H) 6.92(t, J=7.07 Hz, 1H) 3.62-3.73 (m, 2H) 2.46-2.52 (m, 2H) 2.32 (s, 6H)1.59-1.91 (m, 4H). MS [M+1]=389.

Example 11

N-[2-(1-methylpyrrolidin-2-yl)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 11). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 2-(2-aminoethyl)-1-methylpyrrolidine (22 mg, 25 μL, 0.17 mmol)according to Method A to giveN-[2-(1-methylpyrrolidin-2-yl)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(18 mg, 0.045 mmol, 26% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 11.59 (br. s., 1H) 9.10 (s, 1H) 8.98 (dd, J=7.33, 1.77Hz, 1H) 8.70 (dd, J=6.82, 1.77 Hz, 1H) 8.28 (s, 1H) 8.11 (t, J=7.71 Hz,2H) 7.70 (dd, J=8.59, 7.33 Hz, 1H) 7.56-7.62 (m, 1H) 6.92 (t, J=7.20 Hz,1H) 3.66-3.88 (m, 1H) 3.26 (m, 2H) 3.16 (m, 1H) 2.94 (br. s., 3H)2.86-2.92 (m, 1H) 2.83 (d, J=4.04 Hz, 1H) 2.46-2.60 (m, 2H) 2.20-2.36(m, 2H) 2.05-2.20 (m, 2H). MS [M+1]=401.

Example 12

N-[3-(dimethylamino)propyl]-N-methyl-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 12). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N,N,N′-trimethyl-1,3-propanediamine (25.3 μL, 0.17 mmol) accordingto Method A to giveN-[3-(dimethylamino)propyl]-N-methyl-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamidehydrochloride (27.7 mg, 0.071 mmol, 41.4% yield) as a yellow solid. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 9.12 (s, 1H) 8.77 (d, J=7.33 Hz, 1H) 8.32(d, J=6.32 Hz, 2H) 8.10-8.21 (m, 1H) 7.54-7.75 (m, 3H) 6.90-7.01 (m, 1H)3.41 (m, 2H) 3.10 (m, 3H) 2.87-2.93 (m, 8H) 2.13 (m, 2H). MS [M+1]=389.

Example 13

N-(1-methyl-4-piperidyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 13). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 4-amino-1-methylpiperidine (21.6 μL, 0.17 mmol) according to MethodA to giveN-(1-methyl-4-piperidyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(32.4 mg, 0.084 mmol, 48.7% yield) as an orange solid. ¹H NMR (400 MHz,CDCl₃) δ ppm 11.55 (d, J=8.34 Hz, 2H) 9.12 (s, 1H) 8.97 (dd, J=7.33,1.77 Hz, 1H) 8.75 (dd, J=6.82, 1.77 Hz, 1H) 8.22 (s, 1H) 8.13 (d, J=8.84Hz, 1H) 8.03 (d, J=8.59 Hz, 1H) 7.68-7.72 (m, 1H) 7.56-7.61 (m, 1H) 6.92(t, J=7.07 Hz, 1H) 4.19 (br. s., 1H) 2.96 (br. s., 2H) 2.43 (s, 3H) 2.33(br. s., 2H) 2.21 (d, J=9.60 Hz, 2H) 1.85-1.95 (m, 2H). MS [M+1]=387.

Example 14

N-(azetidin-3-yl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 14). A solution of compound 15 (76 mg, 0.17 mmol) inchloroform (1 mL) was treated with trifluoroacetic acid (1.5 mL, 19.6mmol) at room temperature for 24 h (for convenience). The solvents wereremoved in vacuo to giveN-(azetidin-3-yl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamidetrifluoroacetate (76.1 mg, 0.17 mmol, 99% yield) as a yellow solid. ¹HNMR (400 MHz, CDCl₃) δ ppm 9.14-9.20 (m, 1H) 9.10 (d, J=5.81 Hz, 1H)8.67-8.74 (m, 1H) 8.46 (s, 1H) 8.20-8.27 (m, 1H) 8.12 (s, 1H) 7.92 (s,1H) 7.75 (d, J=7.83 Hz, 1H) 7.65 (d, J=6.82 Hz, 1H) 7.11 (t, J=7.20 Hz,1H) 4.67 (dd, J=7.07, 4.55 Hz, 1H) 4.53 (d, J=7.33 Hz, 2H) 2.83 (br.s.,3H). MS [M+1]=345.

Example 15

Tert-butyl3-[(12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carbonyl)amino]azetidine-1-carboxylate(compound 15). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 1-Boc-3-(amino)azetidine (29.7 mg, 0.17 mmol) according to Method Ato give tert-butyl3-[(12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carbonyl)amino]azetidine-1-carboxylate(76 mg, 0.17 mmol, 99% yield) as a yellow-orange solid. ¹H NMR (400 MHz,CDCl₃) δ ppm 11.98 (br. s., 1H) 9.15 (s, 1H) 9.00 (dd, J=7.33, 1.77 Hz,1H) 8.73 (dd, J=7.07, 1.77 Hz, 1H) 8.25 (s, 1H) 8.13 (dd, J=17.56, 7.71Hz, 2H) 7.70-7.76 (m, 1H) 7.58-7.64 (m, 1H) 6.91-6.95 (m, 1H) 4.90 (d,J=6.32 Hz, 1H) 4.46-4.53 (m, 2H) 4.11 (br. s., 2H) 1.52 (s, 9H). MS[M+1]=445.

Example 16

4-(4-isopropylpiperazine-1-carbonyl)-12H-benzo[g]pyrido[2,1-b]quinazoline-12-one(compound 16). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 1-isopropylpiperazine (26.4 μL, 0.17 mmol) according to Method A togive4-(4-isopropylpiperazine-1-carbonyl)-12H-benzo[g]pyrido[2,1-b]quinazoline-12-one(40.9 mg, 0.102 mmol, 59.4% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 9.11-9.14 (m, 1H) 8.79-8.82 (m, 1H) 8.30 (s, 1H)8.10-8.15 (m, 1H) 7.99-8.03 (m, 1H) 7.62-7.68 (m, 1H) 7.53-7.59 (m, 1H)7.45 (dd, J=6.57, 1.52 Hz, 1H) 6.73-6.79 (m, 1H) 4.10 (m, 2H) 3.87 (m,2H) 3.48 (m, 1H) 3.36 (m, 1H) 2.74-2.92 (m, 1H) 2.68 (m, 1H) 2.40 (m,1H) 1.10 (d, J=6.57 Hz, 6H). MS [M+1]=401.

Example 17

N-methyl-N-(1-methylpyrrolidin-3-yl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 17). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N,N′-dimethyl-3-aminopyrrolidine (19.7 mg, 0.17 mmol) according toMethod A to giveN-methyl-N-(1-methylpyrrolidin-3-yl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamidetrifluoroacetate (6 mg, 0.016 mmol, 9.0% yield) as a yellow-orangesolid. ¹H NMR (400 MHz, MeOD) δ ppm 9.11 (s, 1H) 8.83-8.92 (m, 1H)8.24-8.33 (m, 1H) 8.19 (d, J=8.08 Hz, 1H) 8.07 (d, J=8.34 Hz, 1H) 7.92(s, 1H) 7.55-7.74 (m, 3H) 6.95 (q, J=7.33 Hz, 1H) 4.60 (d, J=9.35 Hz,1H) 4.25 (d, J=11.87 Hz, 1H) 4.02-4.14 (m, 1H) 3.47-3.62 (m, 1H)3.17-3.28 (m, 4H) 3.07 (m, 3H) 2.67-2.85 (m, 2H). MS [M+1]=387.

Example 18

4-[(3S)-3-(dimethylamino)pyrrolidine-1-carbonyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-12-one(compound 18). This compound was synthesized from 3a (50 mg, 0.17 mmol)and (3S)-(+3-(dimethylamino)pyrrolidine (19.7 mg, 0.17 mmol) accordingto Method A to give4-[(3S)-3-(dimethylamino)pyrrolidine-1-carbonyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-12-onehydrochloride (34.1 mg, 0.088 mmol, 51.2% yield) as a yellow-orangesolid. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.96 (br. s., 2H) 9.12 (s, 1H) 8.82(td, J=6.63, 1.64 Hz, 1H) 8.31-8.35 (m, 1H) 8.12 (d, J=8.59 Hz, 1H) 8.03(d, J=8.08 Hz, 1H) 7.62-7.68 (m, 1H) 7.53-7.58 (m, 1H) 6.74-6.80 (m, 1H)3.47 (m, 2H) 2.91 (m, 2H) 2.43-2.49 (m, 1H) 2.39 (s, 3H) 2.24-2.33 (m,1H) 2.20 (s, 3H) 2.11 (m, 1H). MS [M+1]=387.

Example 19

4-[3-(diethylamino)pyrrolidine-1-carbonyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-12-one(compound 19). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 3-(diethylamino)pyrrolidine (24.5 mg, 0.17 mmol) according to MethodA to give4-[3-(diethylamino)pyrrolidine-1-carbonyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-12-one(54.7 mg, 0.13 mmol, 76.6% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 9.12 (s, 1H) 8.82 (dd, J=7.58, 1.52 Hz, 1H) 8.28-8.37(m, 2H) 8.12 (d, J=8.59 Hz, 1H) 7.65-7.74 (m, 2H) 7.57-7.63 (m, 1H)6.97-7.02 (m, 1H) 4.10 (m, 1H) 3.97 (m, 1H) 3.81-3.93 (m, 1H) 3.63 (m1H) 3.38 (d, J=7.33 Hz, 1H) 3.14 (m, 1H) 2.87 (m, 1H) 2.33 (br. s., 1H)2.26 (br. s., 1H) 1.33-1.38 (m, 1H) 1.26-1.33 (m, 1H) 1.14 (t, J=7.07Hz, 3H) 1.01 (t, J=7.07 Hz, 3H). MS [M+1]=415.

Example 20

N-[2-(diethylamino)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 20). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N′,N′-diethylethane-1,2-diamine (24.3 μL, 0.17 mmol) according toMethod A to giveN-[2-(diethylamino)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(18.2 mg, 0.047 mmol, 27% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 11.56-11.65 (m, 1H) 9.12 (s, 1H) 8.94-8.99 (m, 1H)8.75 (d, J=7.07 Hz, 1H) 8.41 (s, 1H) 8.14 (d, J=8.08 Hz, 1H) 8.01 (d,J=7.83 Hz, 1H) 7.68 (t, J=7.58 Hz, 1H) 7.51-7.62 (m, 1H) 6.91 (t, J=7.07Hz, 1H) 3.77 (br. s., 2H) 2.83 (m, 6H) 1.21 (t, J=7.20 Hz, 6H). MS[M+1]=389.

Example 21

N-[2-(diisopropylamino)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 21). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N,N-diisopropylethylenediamine (29.9 μL, 0.17 mmol) according toMethod A to giveN-[2-(diisopropylamino)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(33 mg, 0.079 mmol, 46% yield) as an orange solid. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 11.07 (br. s., 1H) 9.11 (s, 1H) 8.92 (d, J=7.33 Hz, 1H)8.58 (d, J=6.32 Hz, 1H) 8.42 (s, 1H) 8.31 (d, J=7.83 Hz, 1H) 8.09 (d,J=7.83 Hz, 1H) 7.73 (t, J=7.07 Hz, 1H) 7.57-7.66 (m, 1H) 7.05 (t, J=7.33Hz, 1H) 3.50 (d, J=5.56 Hz, 2H) 3.13-3.23 (m, 2H) 2.66-2.75 (m, 2H) 1.07(d, J=6.57 Hz, 12H). ¹H NMR (400 MHz, CDCl₃) δ ppm 11.33 (br. s., 1H)9.12 (s, 1H) 8.96 (d, J=6.06 Hz, 1H) 8.76 (d, J=6.57 Hz, 1H) 8.36 (s,1H) 8.13 (d, J=8.08 Hz, 1H) 8.01 (d, J=9.09 Hz, 1H) 7.63-7.72 (m, 1H)7.52-7.61 (m, 1H) 6.90 (t, J=7.07 Hz, 1H) 3.66 (d, J=5.56 Hz, 2H) 3.22(d, J=6.57 Hz, 2H) 2.81 (br. s., 2H) 1.14 (d, J=6.32 Hz, 12H). MS[M+1]=417.

Example 22

12-oxo-N-(2-pyrrolidin-1-ylethyl)-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 22). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 2-pyrrolidin-1-ylethanamine (21.8 μL, 0.17 mmol) according to MethodA to give12-oxo-N-(2-pyrrolidin-1-ylethyl)-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(32.4 mg, 0.084 mmol, 48.7% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl3) δ ppm 11.65 (br. s., 1H) 9.11 (s, 1H) 8.96 (dd, J=7.20, 1.64Hz, 1H) 8.71-8.77 (m, 1H) 8.37 (s, 1H) 8.13 (d, J=8.34 Hz, 1H) 8.02 (d,J=7.83 Hz, 1H) 7.64-7.72 (m, 1H) 7.54-7.63 (m, 1H) 6.87-6.94 (m, 1H)3.79-3.88 (m, 2H) 2.96-3.03 (m, 2H) 2.89 (m, 2H) 2.78 (m, 2H) 2.00 (m,4H). MS [M+1]=387.

Example 23

12-oxo-N-[2-(1-piperidyl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 23). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 1-(2-aminoethyl)piperidine (24.6 μL, 0.17 mmol) according to MethodA to give12-oxo-N-[2-(1-piperidyl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(25 mg, 0.062 mmol, 36.2% yield) as an orange solid. ¹H NMR (400 MHz,CDCl3) δ ppm 11.48 (br. s., 1H) 9.12 (s, 1H) 8.96 (dd, J=7.33, 1.77 Hz,1H) 8.74 (dd, J=6.95, 1.64 Hz, 1H) 8.44 (s, 1H) 8.13 (d, J=8.34 Hz, 1H)8.00 (d, J=8.34 Hz, 1H) 7.68 (dd, J=8.08, 7.07 Hz, 1H) 7.53-7.61 (m, 1H)6.90 (t, J=7.07 Hz, 1H) 3.75 (q, J=5.81 Hz, 2H) 2.73 (t, J=6.19 Hz, 2H)2.61 (br. s., 4H) 1.78 (dt, J=11.24, 5.75 Hz, 4H) 1.54-1.59 (m, 2H). MS[M+1]=401.

Example 24

N-(2-morpholinoethyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 24). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N-(2-aminoethyl)morpholine (22.6 μL, 0.17 mmol) according to MethodA to giveN-(2-morpholinoethyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(28.7 mg, 0.071 mmol, 41.4% yield) as an orange solid. ¹H NMR (400 MHz,DMSO-d6) δ ppm 11.50 (br. s., 1H) 9.12 (s, 1H) 8.96 (dd, J=7.33, 1.77Hz, 1H) 8.75 (dd, J=6.82, 1.77 Hz, 1H) 8.38 (s, 1H) 8.13 (d, J=8.59 Hz,1H) 8.00 (d, J=8.34 Hz, 1H) 7.64-7.73 (m, 1H) 7.54-7.62 (m, 1H) 6.90 (t,J=7.07 Hz, 1H) 3.88-3.97 (m, 4H) 3.77 (q, J=5.89 Hz, 2H) 2.78 (t, J=6.19Hz, 2H) 2.68 (br. s., 4H). MS [M+1]=403.

Example 25

N-[2-(4-methylpiperazin-1-yl)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 25). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 2-(4-methylpiperazin-1-yl)ethanamine (27.6 μL, 0.17 mmol) accordingto Method A to giveN-[2-(4-methylpiperazin-1-yl)ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(11.7 mg, 0.028 mmol, 16.3% yield) as a yellow-orange solid. ¹H NMR (400MHz, CDCl₃) δ ppm 11.46 (br. s., 1H) 9.14 (s, 1H) 8.98 (dd, J=7.33, 1.77Hz, 1H) 8.76 (dd, J=6.82, 1.77 Hz, 1H) 8.43 (s, 1H) 8.16 (d, J=8.59 Hz,1H) 8.08 (d, J=8.34 Hz, 1H) 7.65-7.73 (m, 1H) 7.60 (dd, J=6.82, 1.26 Hz,1H) 6.92 (t, J=7.07 Hz, 1H) 3.74-3.83 (m, 2H) 2.60-2.88 (m, 10H) 2.38(s, 3H). MS [M+1]=416.

Example 26

N-[2-[2-methoxyethyl(methyl)amino]ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 26). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N′-(2-methoxyethyl)-N′-methyl-ethane-1,2-diamine (27.3 mg, 0.21mmol) according to Method A to giveN-[2-[2-methoxyethyl(methyl)amino]ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamidetrifluoroacetate (15 mg, 0.037 mmol, 21.5% yield) as an orange gum. ¹HNMR (400 MHz, CDCl₃) δ ppm 11.79 (br. s., 1H) 9.10 (s, 2H) 9.00 (dd,J=7.33, 1.52 Hz, 1H) 8.68-8.73 (m, 1H) 8.43 (s, 1H) 8.10 (dd, J=13.64,8.34 Hz, 3H) 7.70 (t, J=7.58 Hz, 2H) 7.56-7.62 (m, 2H) 6.94 (t, J=7.20Hz, 1H) 4.07 (br. s., 2H) 3.80 (t, J=4.55 Hz, 2H) 3.68 (br. s., 1H) 3.55(br. s., 2H) 3.36-3.41 (m, 4H) 3.07 (s, 3H). ¹⁹F NMR (376 MHz, CDCl₃) δppm −170.87 (br. s., 3 F). MS [M+1]=405.

Example 27

N-[2-(dimethylamino)-1-methyl-ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 27). This compound was synthesized from 3a (50 mg, 0.17 mmol)and N1,N1-dimethylpropane-1,2-diamine (22.2 μL, 0.17 mmol) according toMethod A to giveN-[2-(dimethylamino)-1-methyl-ethyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamidehydrochloride (38.7 mg, 0.103 mmol, 60% yield) as a yellow-orange solid.¹H NMR (400 MHz, CDCl₃) δ ppm 9.12 (s, 1H) 8.82 (td, J=6.63, 1.64 Hz,1H) 8.31-8.35 (m, 1H) 8.12 (d, J=8.59 Hz, 1H) 8.03 (d, J=8.08 Hz, 1H)7.62-7.68 (m, 1H) 7.53-7.58 (m, 1H) 6.74-6.80 (m, 1H) 3.47 (br. s., 1H)2.91 (br. s., 1H) 2.43-2.49 (m, 1H) 2.39 (s, 3H) 2.20 (s, 3H) 1.96 (br.s., 3H). MS [M+1]=375.

Example 28

N-[2-(dimethylamino)propy]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 28). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 2-(dimethylamino)propan-1-amine (21.1 mg, 0.21 mmol) according toMethod A to giveN-[2-(dimethylamino)propyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(52 mg, 0.139 mmol, 80.6% yield) as a yellow solid. ¹H NMR (400 MHz,CDCl₃) δ ppm 11.70 (br. s., 1H) 9.09 (s, 1H) 8.94 (dd, J=7.33, 1.77 Hz,1H) 8.73 (dd, J=6.82, 1.77 Hz, 1H) 8.27 (s, 1H) 8.12 (d, J=8.59 Hz, 1H)7.99 (d, J=8.59 Hz, 1H) 7.66 (dd, J=7.96, 6.95 Hz, 1H) 7.53-7.58 (m, 1H)6.89 (t, J=7.07 Hz, 1H) 3.77 (dt, J=14.02, 5.62 Hz, 1H) 3.40-3.48 (m,1H) 2.95-3.05 (m, 1H) 2.51 (s, 6H) 1.15 (d, J=6.32 Hz, 3H). MS[M+1]=375.

Example 29

N-(1-methylazetidin-3-yl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 29). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 1-methylazetidin-3-amine dihydrochloride (28.4 uL, 0.26 mmol)according to Method A to give crude product, which was purified byautomated normal-phase chromatography (0-20% MeOH/DCM, 4 g silica gelcartridge) to giveN-(1-methylazetidin-3-yl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(32.8 mg, 0.092 mmol, 53.1% yield) as a red-orange glass. 1H NMR (400MHz, DMSO-d6) δ ppm 11.45 (d, J=6.57 Hz, 1H) 9.11 (s, 1H) 8.93 (dd,J=7.33, 1.77 Hz, 1H) 8.50 (dd, J=6.82, 1.77 Hz, 1H) 8.42 (s, 1H) 8.31(d, J=8.59 Hz, 1H) 8.20 (d, J=8.59 Hz, 1H) 7.71-7.76 (m, 1H) 7.59-7.64(m, 1H) 7.05 (t, J=7.07 Hz, 1H) 4.54-4.63 (m, 1H) 3.84 (t, J=7.83 Hz,2H) 3.35-3.38 (m, 2H) 2.47 (s, 3H) MS [M+1]=359.

Example 30

N-(2-amino-2-methyl-propyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 30). This compound was synthesized from 3a (100 mg, 0.34 mmol)and 2-methylpropane-1,2-diamine (106 uL, 1.03 mmol) according to MethodA to giveN-(2-amino-2-methyl-propyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(110 mg, 0.31 mmol, 88.6% yield) as an orange solid. 1H NMR (400 MHz,DMSO-d6) δ ppm 11.38 (t, J=5.31 Hz, 1H) 9.12 (s, 1H) 8.92 (dd, J=7.33,1.77 Hz, 1H) 8.54 (dd, J=6.95, 1.64 Hz, 1H) 8.38 (s, 1H) 8.31 (d, J=8.34Hz, 1H) 8.13 (d, J=8.59 Hz, 1H) 7.73 (dd, J=8.34, 6.82 Hz, 1H) 7.58-7.63(m, 1H) 7.06 (t, J=7.07 Hz, 1H) 3.34-3.40 (m, 2H) 2.69 (s, 2H) 1.21 (s,6H) MS [M+1]=361.

Example 31

Method B: Bis-methylation of primary amine.N-[2-(dimethylamino)-2-methyl-propyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 31). To a solution of compound 30 (77 mg, 0.21 mmol) in MeCN(1.5 mL) and trimethyl orthoformate (0.50 mL) was added formaldehyde(128 uL, 2.14 mmol), sodium cyanoborohydride (85 mg, 2.14 mmol) and onedrop of glacial acetic acid. The contents were stirred at roomtemperature for 4 h, then treated with 1M NaOH, taken up in EtOAc,washed with 10% Na2CO3 (3×), brine, dried over MgSO₄, filtered and thesolvent removed in vacuo to give an orange residue. This material waspurified by automated normal-phase chromatography (0-20% MeOH/DCM, 4 gsilica gel cartridge) to giveN-[2-(dimethylamino)-2-methyl-propyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(45 mg, 0.12 mmol, 54% yield) as an orange solid. 1H NMR (400 MHz,CDCl3) δ ppm 11.87 (br. s., 1H) 9.11 (s, 1H) 8.95 (dd, J=7.33, 1.77 Hz,1H) 8.73 (dd, J=7.07, 1.77 Hz, 1H) 8.29 (s, 1H) 8.13 (d, J=8.34 Hz, 1H)8.00 (d, J=8.34 Hz, 1H) 7.67 (ddd, J=8.27, 6.88, 1.26 Hz, 1H) 7.56 (ddd,J=8.21, 6.82, 1.14 Hz, 1H) 6.90 (t, J=7.07 Hz, 1H) 3.63 (d, J=4.80 Hz,2H) 2.56 (s, 6H) 1.28 (s, 6H) MS [M+1]=389.

Example 32

N-(2-aminopropyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 32). This compound was synthesized from 3a (100 mg, 0.17 mmol)and 1,2-diaminopropane (88 uL, 1.03 mmol) according to Method A to givecrude product, which was purified by automated normal-phasechromatography (0-20% MeOH/DCM, 4 g silica gel cartridge) to giveN-(2-aminopropyl)-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(23.9 mg, 0.069 mmol, 20% yield) as an orange solid. 1H NMR (400 MHz,DMSO-d6) δ ppm 11.27 (br. s., 1H) 9.11 (s, 1H) 8.92 (dd, J=7.20, 1.64Hz, 1H) 8.52 (dd, J=6.82, 1.77 Hz, 1H) 8.43 (s, 1H) 8.31 (d, J=8.59 Hz,1H) 8.14 (d, J=8.59 Hz, 1H) 7.73 (t, J=7.71 Hz, 1H) 7.58-7.63 (m, 1H)7.06 (t, J=7.07 Hz, 1H) 3.42-3.51 (m, 1H) 3.35-3.39 (m, 1H) 3.20 (dd,J=12.51, 5.94 Hz, 1H) 1.19 (d, J=6.57 Hz, 3H) MS [M+1]=347.

Example 33

N-[(2R)-2-aminopropyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 33). This compound was synthesized from 3a (100 mg, 0.17 mmol)and (2R)-propane-1,2-diamine dihydrochloride (152 mg, 1.03 mmol)according to Method A to give crude product, which was purified byautomated normal-phase chromatography (0-30% MeOH/DCM, 4 g silica gelcartridge) to giveN-[(2R)-2-aminopropyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(42.2 mg, 0.122 mmol, 35% yield) as a yellow-orange solid. 1H NMR (400MHz, CDCl3) δ ppm 11.57 (br. s., 1H) 9.12 (s, 1H) 8.97 (dd, J=7.20, 1.64Hz, 1H) 8.75 (dd, J=6.82, 1.52 Hz, 1H) 8.28 (s, 1H) 8.12 (d, J=8.08 Hz,1H) 8.03 (d, J=8.59 Hz, 1H) 7.67 (dd, J=8.21, 6.95 Hz, 1H) 7.54-7.60 (m,1H) 6.91 (t, J=7.07 Hz, 1H) 3.71 (dt, J=13.14, 5.31 Hz, 1H) 3.33-3.48(m, 2H) 1.31 (d, J=6.32 Hz, 3H) MS [M+1]=347.

Example 34

N-[(2R)-2-(dimethylamino)propyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 34). This compound was synthesized from compound 33 (35 mg,0.10 mmol) according to Method B to give crude product, which waspurified by automated normal-phase chromatography (0-20% MeOH/DCM, 4 gsilica gel cartridge) to giveN-[(2R)-2-aminopropyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(22.3 mg, 0.060 mmol, 59% yield) as a yellow-orange solid. 1H NMR (400MHz, CDCl3) δ ppm 11.73 (br. s., 1H) 9.11 (s, 1H) 8.95 (dd, J=7.33, 1.77Hz, 1H) 8.73 (dd, J=7.07, 1.77 Hz, 1H) 8.29 (s, 1H) 8.12 (d, J=8.34 Hz,1H) 8.01 (d, J=8.08 Hz, 1H) 7.67 (ddd, J=8.34, 6.82, 1.26 Hz, 1H) 7.56(ddd, J=8.27, 6.88, 1.26 Hz, 1H) 6.89 (t, J=7.07 Hz, 1H) 3.74-3.81 (m,1H) 3.47 (ddd, J=14.08, 8.78, 3.41 Hz, 1H) 3.00-3.08 (m, 1H) 2.53 (s,6H) 1.17 (d, J=6.57 Hz, 3H) MS [M+1]=375.

Example 35

N-[(2S)-2-aminopropyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 35). This compound was synthesized from 3a (100 mg, 0.17 mmol)and (2R)-propane-1,2-diamine dihydrochloride (152 mg, 1.03 mmol)according to Method A to give crude product, which was purified byautomated normal-phase chromatography (0-30% MeOH/DCM, 4 g silica gelcartridge) to giveN-[(25)-2-aminopropyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(33.1 mg, 0.096 mmol, 28% yield) as a yellow-orange solid. 1H NMR (400MHz, CDCl3) δ ppm 11.57 (br. s., 1H) 9.12 (s, 1H) 8.97 (dd, J=7.33, 1.77Hz, 1H) 8.75 (dd, J=6.82, 1.77 Hz, 1H) 8.28 (s, 1H) 8.13 (d, J=8.08 Hz,1H) 8.03 (d, J=8.34 Hz, 1H) 7.67 (ddd, J=8.27, 6.88, 1.26 Hz, 1H)7.54-7.59 (m, 1H) 6.91 (t, J=7.20 Hz, 1H) 3.71 (ddd, J=13.07, 5.87, 4.55Hz, 1H) 3.33-3.51 (m, 2H) 1.31 (d, J=6.32 Hz, 3H) MS [M+1]=347.

Example 36

N-[(2S)-2-(dimethylamino)propyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 36). This compound was synthesized from compound 35 (26.5 mg,0.080 mmol) according to Method B to give crude product, which waspurified by automated normal-phase chromatography (0-20% MeOH/DCM, 4 gsilica gel cartridge) to giveN-[(2S)-2-aminopropyl]-12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(19 mg, 0.051 mmol, 66% yield) as a yellow-orange solid. 1H NMR (400MHz, CDCl3) δ ppm 11.71 (br. s., 1H) 9.11 (s, 1H) 8.94 (dd, J=7.33, 1.77Hz, 1H) 8.73 (dd, J=7.07, 1.77 Hz, 1H) 8.28 (s, 1H) 8.12 (d, J=8.08 Hz,1H) 8.00 (d, J=8.08 Hz, 1H) 7.67 (ddd, J=8.27, 6.88, 1.26 Hz, 1H) 7.56(ddd, J=8.21, 6.82, 1.14 Hz, 1H) 6.89 (t, J=7.07 Hz, 1H) 3.77 (ddd,J=14.02, 6.06, 5.18 Hz, 1H) 3.45 (ddd, J=13.96, 8.91, 3.41 Hz, 1H)2.97-3.06 (m, 1H) 2.52 (s, 6H) 1.16 (d, J=6.57 Hz, 3H) MS [M+1]=375.

Example 37

12-oxo-N-[2-(4-pyridyl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(compound 37). This compound was synthesized from 3a (50 mg, 0.17 mmol)and 4-(2-aminoethyl)pyridine (20.8 μL, 0.17 mmol) according to Method Ato give12-oxo-N-[2-(4-pyridyl)ethyl]-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide(43.5 mg, 0.11 mmol, 64% yield) as an orange solid. ¹H NMR (400 MHz,CDCl₃) δ ppm 11.42 (br. s., 1H) 9.09 (s, 1H) 8.97 (dd, J=7.33, 1.77 Hz,1H) 8.76 (dd, J=7.07, 1.77 Hz, 1H) 8.62-8.67 (m, 2H) 8.09 (dd, J=17.81,8.46 Hz, 2H) 7.66-7.74 (m, 1H) 7.54-7.62 (m, 1H) 7.48 (s, 1H) 7.37 (d,J=6.06 Hz, 2H) 6.92 (t, J=7.07 Hz, 1H) 3.97-4.08 (m, 2H) 3.05-3.15 (m,2H). MS [M+1]=395.

Example 38

2-(dimethylamino)ethyl12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxylate. This compoundwas synthesized from 3a (50 mg, 0.17 mmol) and 2-dimethylaminoethanol(41.6 μL, 0.41 mmol) according to Method A to give2-(dimethylamino)ethyl12-oxo-12H-benzo[g]pyrido[2,1-b]quinazoline-4-carboxylate (47 mg, 0.13mmol, 47.2% yield) as an orange solid. ¹H NMR (400 MHz, CDCl₃) δ ppm9.10 (s, 1H) 8.87 (dd, J=7.45, 1.64 Hz, 1H) 8.36 (s, 1H) 8.11 (d, J=8.34Hz, 1H) 8.02 (d, J=8.34 Hz, 1H) 7.78 (dd, J=6.69, 1.64 Hz, 1H) 7.64(ddd, J=8.27, 6.76, 1.14 Hz, 1H) 7.52-7.57 (m, 1H) 6.72 (dd, J=7.33,6.57 Hz, 1H) 4.58 (t, J=5.81 Hz, 2H) 2.80 (t, J=5.68 Hz, 2H) 2.38 (s,6H). MS [M+1]=362.

As the data herein indicate, a broad variety of compounds of formula Iwere found effective at low concentrations. IC₅₀ values for exemplarycompounds of formula I (see above for compound names and structures) areprovided in Table 1 below.

TABLE 1 Compounds and their efficacies ID RPA194 (IC₅₀ μM) NCL (IC₅₀ μM)Compound 1 22 NT Compound 2 >100 >100 Compound 3 1 26 Compound 4 16 >100Compound 5 12 >100 Compound 6 13 2 Compound 7 32 >100 Compound 8 5 >100Compound 9 0.43 0.48 Compound 10 0.66 1.53 Compound 11 2.0 2.6 Compound12 33 >100 Compound 13 0.18 1.38 Compound 14 0.21 0.18 Compound 1535 >100 Compound 16 >100 >100 Compound 17 42 >100 Compound 18 >100 >100Compound 19 32 11 Compound 20 0.14 0.19 Compound 21 3.0 1.7 Compound 220.09 0.08 Compound 23 0.18 0.08 Compound 24 1.3 0.60 Compound 25 1.1 1.8Compound 26 0.73 0.70 Compound 27 0.11 0.16 Compound 28 0.04 0.04Compound 29 0.90 0.47 Compound 30 0.27 0.20 Compound 31 0.16 0.14Compound 32 0.10 0.10 Compound 33 0.11 0.81 Compound 34 0.26 0.05Compound 35 0.12 0.17 Compound 36 0.04 0.04 Compound 37 3.5 >100

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method for modulating RNA Pol I activity in a mammalian cell or apopulation of mammalian cells, the method comprising contacting the cellor the population of cells with a compound of formula I:

wherein X is NR₂; wherein L is R₃ or an optionally substitutedcycloamine

wherein R₁ is a straight-chained or branched C₁-C₆ hydrocarbon group(e.g., an alkyl group, an alkenyl group, an alkynyl group, alkylolgroup, hydroxyalkyl group, alkoxy group, alkoxyalkyl group, cyclicgroups, whether substituted or unsubstituted, such as cyclopentyl,cyclohexyl, pyramido, phenyl, or benzyl, cycloalkyl, heterocyclyl,indole, wherein each of alkyl, aryl, or heterocyclyl moiety may beunsubstituted or substituted with one or more substituents selected fromthe group consisting of halo, hydroxy, carboxy, phosphoryl, phosphonyl,phosphono C₁-C₆ alkyl, carboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkyl,dicarboxy halo C₁-C₆ alkyl, sulfonyl, cyano, nitro, alkoxy, alkylthio,acyl, acyloxy, thioacyl, acylthio, aryloxy, amino, alkylamino,dialkylamino, trialkylamino, arylalkylamino, guanidino, aldehydo,ureido, and aminocarbonyl, a branched or straight-chain alkylamino,dialkylamino, or alkyl or dialkylaminoalkyl, or thioalkyl, thioalkenyl,thioalkynyl, aryloxy, acyloxy, thioacyl, amido, sulphonamido, etc.), orthe like; when X is NR₂, R₂ is H or a straight-chained C₁-C₆ alkylgroup; when L is R₃, R₃ is a straight-chained or branched C₂-C₆ alkylgroup; when L is

m=1-8 and each Y is independently selected from (CH₂)_(n)Y¹ _(p) whereinn=1-8, p=0-4 and the sum of n and p is at least 2, and each Y¹ isindependently selected from NR₄, O, S, or P, wherein R₄ is ashereinbefore defined for R₃, and X≠O; or a pharmaceutically acceptablesalt, solvate, stereoisomer, or a prodrug thereof.
 2. The method ofclaim 1, wherein L is R₃ and R₃ is a straight-chained or branched C₂-C₆alkyl group having at least one chiral carbon.
 3. The method of claim 1,wherein the compound has formula II,

wherein R₁=H and R₂=C₁-C₆ alkyl, substituted with one or more C₁-C₄alkyl, OH, NH₂, NR₃R₄, cyano, SO₂R₃, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl (including but not limited toimidazolyl, imidazolidinonyl, pyridyl, indolyl, oxazolyl, thiazolyl,oxadiazolyl), substituted or unsubstituted cycloalkyl or substituted orunsubstituted nitrogen-containing heterocycles including, but notlimited to azetidine, pyrrolidine, piperidine, piperazine, azapine,morpholino; wherein R₃ and R₄, are independently selected from the groupincluding H, C₁-C₆ alkyl, and C₁-C₄ alkoxyl alkyl, having at least onechiral carbon, when R₂ is substituted with at least one NR₃R₄ group. 4.The method of claim 3, comprising a chirally pure stereoisomer ofcompound of formula II when R₂ is substituted with at least one NR₃R₄group and a pharmaceutically acceptable carrier.
 5. The method of claim1, wherein the compound is selected from the group consisting of:


6. A method for treating a hyperproliferative disease in a subject, themethod comprising administering an effective amount of a compound offormula I to the subject:

wherein X is NR₂; wherein L is R₃ or an optionally substitutedcycloamine

wherein R₁ is a straight-chained or branched C₁-C₆ hydrocarbon group(e.g., an alkyl group, an alkenyl group, an alkynyl group, alkylolgroup, hydroxyalkyl group, alkoxy group, alkoxyalkyl group, cyclicgroups, whether substituted or unsubstituted, such as cyclopentyl,cyclohexyl, pyramido, phenyl, or benzyl, cycloalkyl, heterocyclyl,indole, wherein each of alkyl, aryl, or heterocyclyl moiety may beunsubstituted or substituted with one or more substituents selected fromthe group consisting of halo, hydroxy, carboxy, phosphoryl, phosphonyl,phosphono C₁-C₆ alkyl, carboxy C₁-C₆ alkyl, dicarboxy C₁-C₆ alkyl,dicarboxy halo C₁-C₆ alkyl, sulfonyl, cyano, nitro, alkoxy, alkylthio,acyl, acyloxy, thioacyl, acylthio, aryloxy, amino, alkylamino, dialkylamino, trialkylamino, arylalkylamino, guanidino, aldehydo, ureido,and aminocarbonyl, a branched or straight-chain alkylamino,dialkylamino, or alkyl or dialkylaminoalkyl, or thioalkyl, thioalkenyl,thioalkynyl, aryloxy, acyloxy, thioacyl, amido, sulphonamido, etc.), orthe like; when X is NR₂, R₂ is H or a straight-chained C₁-C₆ alkylgroup; when L is R₃, R₃ is a straight-chained or branched C₂-C₆ alkylgroup; when L is

m=1-8 and each Y is independently selected from (CH₂)_(n)Y¹ _(p) whereinn=1-8, p=0-4 and the sum of n and p is at least 2, and each Y¹ isindependently selected from NR₄, O, S, or P, wherein R₄ is ashereinbefore defined for R₃, and X≠O; or a pharmaceutically acceptablesalt, solvate, stereoisomer, or a prodrug thereof.
 7. The method ofclaim 6, wherein L is R₃ and R₃ is a straight-chained or branched C₂-C₆alkyl group having at least one chiral carbon.
 8. The method of claim 6,wherein the compound has formula II,

wherein R₁=H and R₂=C₁-C₆ alkyl, substituted with one or more C₁-C₄alkyl, OH, NH₂, NR₃R₄, cyano, SO₂R₃, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl (including but not limited toimidazolyl, imidazolidinonyl, pyridyl, indolyl, oxazolyl, thiazolyl,oxadiazolyl), substituted or unsubstituted cycloalkyl or substituted orunsubstituted nitrogen-containing heterocycles including, but notlimited to azetidine, pyrrolidine, piperidine, piperazine, azapine,morpholino; wherein R₃ and R₄, are independently selected from the groupincluding H, C₁-C₆ alkyl, and C₁-C₄ alkoxyl alkyl, having at least onechiral carbon, when R₂ is substituted with at least one NR₃R₄ group. 9.The method of claim 8, comprising a chirally pure stereoisomer ofcompound of formula II when R₂ is substituted with at least one NR₃R₄group and a pharmaceutically acceptable carrier.
 10. The method of claim6, wherein the compound selected from the group consisting of


11. The method of claim 6, wherein the hyperproliferative disease iscancer.
 12. The method of claim 11, further comprising administering tothe subject at least one other biologically active agent.